Dear colleagues,
I am wondering how to interprete titration (attached) of my soluble protein (50 kDa) with its highly polar LMW ligand (amide and sulfonic acid groups, 1.4 kDa). TItration was performed in PBS, protein in cell was 60uM and ligand in syringe was 1mM, with 20 injections (2uL, 4s), on Auto-iTC200.
Thank you in advance for any comments, suggestions.
Hello @Jakub Began: I cannot provide an evaluation of the ITC data without seeing the raw ITC data, before any adjustment for integration. It looks like the 1st few injections are not having the heat change return completely to baseline.
My first observation is that the initial heat change is endothermic, and the final heat change is exothermic. What does the control titration look like? The control titration is buffer in the ITC cell, and ligand in the syringe. How are the protein and ligand prepared - are they in matched solution? Did you check the pH of the samples to make sure they match?
It is possible that the binding reaction is endothermic, and the heat of dilution is exothermic. Once the data is corrected for heat of dilution, it is possible to fit to a binding curve, though it appears that the heat change at the end of the titration has not fully ended, and also it is not clear if there is one or more processes causing heat change in ITC. It also looks like there are 2-3 binding sites from the data.
I agree with Verna Frasca , " It is possible that the binding reaction is endothermic, and the heat of dilution is exothermic". The latter if you have not subtracted blank and after integrate the heat rate signals.
Hello, curves are already corrected for heat of dilution. Blank was ligand into buffer. I will try to find the raw data befor substraction. Ligand was prepared in buffer taken after protein dialysis, so this should not be problem. On the other hand, pH might changed. I have not checked that. Could the explanation be that the ligand induces some changes in the protein at the very beginning? To get more relevant curve, what experimental setup woul you recommnend?
As Verna Frasca said, you may need to increase the concentration in the syringe in order to better define the laste part of the titration.
Even if you take the appropriate precautions (e.g., dissolve ligand into buffer dialysis) and perform the blank titration, the background heat affect from the injection may be significant. In your case, it looks like the background heat effect is exothermic and there are several biding sites (as already commented) with positive interaction enthalpy. The binding sites seem to be different (there are two inflection points) or they could be identical but showing cooperativity. Different binding processes (or sites) do not necessarily reflect conformational changes.
Is you protein oligomeric (or show a monomer-oligomer equilibrium)?
Do you know how many ligands your protein can bind?
- Badges
- Science topic
- Similar topics
- Philosophy
- Esthetics
- Interpretation